CSB349H1 Lecture Notes - Lecture 8: Xist (Gene), X-Inactivation, Zygosity
14 May 2018
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Sequencing of Smchd1
Wild-Type Gene | Gene Trap Construct (bottom)
[Smchd1]
Same Phenotype via Direct Gene Mutation
Lecture 8(c): Heterochromatin & Positioning Effect
Smchd1 Locus:
• MommeD1 mutation was mapped and found in the Smchd1 locus
o MommeD1 WT: Cytosine (C)
o MommeD1 Homozygous mutation: Thymine (T)
o MommeD1 Heterozygous mutation: (C)/(T)
• Smchd1 has reduced expression in MommeD1 homozygous embryos
Gene Trap Insertion:
• Gene trap insertion was completed to confirm Smchd1 is the candidate gene that is
causative of the MommeD1 phenotype
Complementation:
• Mutagenizing an organism can lead to several mutations; we aim to discover a mutation that
will affect one gene and give rise to a mutant phenotype:
o If mapping is done incorrectly, a mutation can be generated in a different location and
give rise to a mutant phenotype that is not associated with our gene of interest
ð In order to confirm that a gene mutation gave rise to a mutant phenotype, we must be able to
use a WT version of the gene to recover the WT phenotype from a mutant phenotype:
o Recessive mutation:
§ Complete a knock-out experiment to generate a mutant phenotype
§ Artificially insert WT gene which allows the recovery of WT phenotype
Experiment:
• MommeD1 mutation is semi-dominant, therefore, we cannot do the knock-out experiment,
but will complete a gene trap construct:
o Gene trap consists of modifying the WT gene: have a few 100 bps of the gene followed
by an antibiotic resistant gene
§ Smchd1 gene contains a selectable marker and a truncated, nonfunctional
version of the protein
• Reintroduced the gene trap construct into a mouse with the initial GFP construct that was
used previously for the screen and completed a FACS analysis:
o Gene trap construct was capable of achieving the same phenotype of a heterozygous
mutant
ð Developed the same mutation generating the same phenotype with the candidate gene in a
different way which implies it is the gene
Document Summary
Lecture 8(c): heterochromatin & positioning effect: mommed1 mutation was mapped and found in the smchd1 locus, mommed1 wt: cytosine (c, mommed1 homozygous mutation: thymine (t, mommed1 heterozygous mutation: (c)/(t) Sequencing of smchd1: smchd1 has reduced expression in mommed1 homozygous embryos. Gene trap insertion: gene trap insertion was completed to confirm smchd1 is the candidate gene that is causative of the mommed1 phenotype. In order to confirm that a gene mutation gave rise to a mutant phenotype, we must be able to use a wt version of the gene to recover the wt phenotype from a mutant phenotype: recessive mutation: Complete a knock-out experiment to generate a mutant phenotype. Artificially insert wt gene which allows the recovery of wt phenotype. Developed the same mutation generating the same phenotype with the candidate gene in a different way which implies it is the gene.
